Lunasin is a redox sensitive intrinsically disordered peptide with two transiently populated α-helical regions

Lunasin is a 43 amino acid peptide with anti-cancer, antioxidant, anti-inflammatory and cholesterol-lowering properties. Although the mechanism of action of lunasin has been characterized to some extent, its exact three-dimensional structure as well as the function of the N-terminal sequence remains unknown. We established a novel method for the production of recombinant lunasin that allows efficient isotope labeling for NMR studies. Initial studies showed that lunasin can exist in a reduced or oxidized state with an intramolecular disulfide bond depending on solution conditions. The structure of both forms of the peptide at pH 3.5 and 6.5 was characterized by CD spectroscopy and multidimensional NMR methods. The data indicate that lunasin belongs to the class of intrinsically disordered proteins. The analysis of secondary structure propensities indicates the presence of two helical regions and an extended (beta strand) conformation at the C-terminus. We hypothesize that the transient secondary structure elements could be stabilized upon interaction with the histones H3 and H4. The newly discovered redox properties of lunasin could explain its antioxidant and anti-inflammatory activity.     

Identification and bioavailability of a chromatin-binding peptide (Lunasin) from Korean Soybean

Lunasin is a novel peptide with great potential as a nontoxic chemopreventive drug. This compound might account for part of the anticancer effects reported from studies with soybean(Glycine max). We studied its isolation, purification and biological assay, and observed that both its band from soybean and one from synthetic lunasin were <5 kD in their molecular weights. Among all the crop varieties tested, only the soybean produced a lunasin band on our western blot. Levels of this peptide ranged from 0.045 mg per gram of seed for the Hanbatkong cultivar to 0.156 mg per gram seed for Poolunkong’. The effect on colony formation by lunasin from different soybean extracts was significantly higher than for either the positive control or the synthetic lunasin. When lunasin was present in those natural extracts, histone acetylation decreased 100% compared with cells that were treated with Na-butyrate.     

Scalable purification and characterization of the anticancer lunasin peptide from soybean

Lunasin is a peptide derived from the soybean 2S albumin seed protein that has both anticancer and anti-inflammatory activities. Large-scale animal studies and human clinical trials to determine the efficacy of lunasin in vivo have been hampered by the cost of synthetic lunasin and the lack of a method for obtaining gram quantities of highly purified lunasin from plant sources. The goal of this study was to develop a large-scale method to generate highly purified lunasin from defatted soy flour. A scalable method was developed that utilizes the sequential application of anion-exchange chromatography, ultrafiltration, and reversed-phase chromatography. This method generates lunasin preparations of >99% purity with a yield of 442 mg/kg defatted soy flour. Mass spectrometry of the purified lunasin revealed that the peptide is 44 amino acids in length and represents the original published sequence of lunasin with an additional C-terminal asparagine residue. Histone-binding assays demonstrated that the biological activity of the purified lunasin was similar to that of synthetic lunasin. This study provides a robust method for purifying commercial-scale quantities of biologically-active lunasin and clearly identifies the predominant form of lunasin in soy flour. This method will greatly facilitate the development of lunasin as a potential nutraceutical or therapeutic anticancer agent.     

Formation of molten globule-like state during acid denaturation of Aspergillus niger glucoamylase

Acid denaturation of Aspergillus niger glucoamylase was studied using different conformational probes. Both far-UV CD spectral signal (MRE_222 nm) and tryptophan fluorescence remained unchanged in the pH range, 7.0–3.0 but decreased significantly below pH 3.0, whereas ANS fluorescence showed a marked increase below pH 1.5. Maximal changes in MRE_222 nm and ANS fluorescence were noticed at pH 1.0. Acid-denatured state of glucoamylase at pH 1.0 retained a significant amount of secondary structure as reflected from far-UV CD spectra but showed a deformed tertiary structure with significant exposure of nonpolar groups as well as tryptophan residues as revealed by increased ANS fluorescence, decreased tryptophan fluorescence and three-dimensional fluorescence spectral signals and increase in K_sv value in acrylamide quenching experiments. Acid-denatured state showed no significant variation in the CD spectral signal throughout the temperature range, 0–100 °C. However, a late cooperative transition was observed upon GdnHCl treatment, compared to the native enzyme. All these results suggested that the acid-denatured state of glucoamylase at pH 1.0 represented the molten globule-like state.     

Additional disulfide bonds in insulin: Prediction,recombinant expression,receptor binding affinity,and stability

The structure of insulin, a glucose homeostasis-controlling hormone, is highly conserved in all vertebrates and stabilized by three disulfide bonds. Recently, we designed a novel insulin analogue containing a fourth disulfide bond located between positions A10-B4. The N-terminus of insulin''s B-chain is flexible and can adapt multiple conformations. We examined how well disulfide bond predictions algorithms could identify disulfide bonds in this region of insulin. In order to identify stable insulin analogues with additional disulfide bonds, which could be expressed, the C_β cut-off distance had to be increased in many instances and single X-ray structures as well as structures from MD simulations had to be used. The analogues that were identified by the algorithm without extensive adjustments of the prediction parameters were more thermally stable as assessed by DSC and CD and expressed in higher yields in comparison to analogues with additional disulfide bonds that were more difficult to predict. In contrast, addition of the fourth disulfide bond rendered all analogues resistant to fibrillation under stress conditions and all stable analogues bound to the insulin receptor with picomolar affinities. Thus activity and fibrillation propensity did not correlate with the results from the prediction algorithm.     

Antioxidant and anti-inflammatory properties of cancer preventive peptide lunasin in RAW 264.7 macrophages

Oxidative stress and inflammation are two of the most critical factors implicated in carcinogenesis and other degenerative disorders. We have investigated how lunasin, a known anti-cancer seed peptide, affect these factors. This peptide inhibits linoleic acid oxidation and acts as 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical scavenger. Furthermore, using LPS-stimulated RAW 264.7 macrophages, we have demonstrated that lunasin reduces, in a significant dose-dependent manner, the production of reactive oxygen species (ROS) by LPS-induced macrophages. Lunasin also inhibits the release of pro-inflammatory cytokines (tumor necrosis factor-α [TNF-α] and interleukine-6 [IL-6]). On the basis of these potent antioxidant and anti-inflammatory properties, we propose lunasin not only as a cancer preventive and therapeutic agent but also as an agent against other inflammatory-related disorders.     

Lunasin Alleviates Allergic Airway Inflammation while Increases Antigen-Specific Tregs

Lunasin is a naturally occurring peptide isolated from soybeans and has been explored in cancer treatment. Lunasin inhibits NF-κB activation and thus pro-inflammatory cytokine and mediator production in macrophages. In this study we demonstrate that lunasin can effectively suppress allergic airway inflammation in two murine models of asthma. In an OVA+Alum sensitization model, intranasal lunasin treatment at the time of OVA challenges significantly reduced total cells counts in bronchoalveolar lavage (BAL) fluid and eosinophilia, peribronchiolar inflammatory infiltration, goblet cell metaplasia and airway IL-4 production. In an OVA+LPS intranasal sensitization model, lunasin treatment either at the time of sensitization or challenge has similar effects in suppress allergic airway inflammation including significantly reduced total cell and eosinophil counts in BAL fluid, inflammatory gene Fizz1 expression in the lung, and IL-4 production by OVA re-stimulated cells from mediastinal lymph nodes. We further show that intranasal instillation of OVA+lunasin significantly increases OVA-specific regulatory T cell (Treg) accumulation in the lung comparing to OVA only treatment. Taken together, our results suggest lunasin as an anti-inflammatory agent can be potentially used in asthma therapy or as an adjuvant to enhance the induction of antigen-specific Tregs and thus boost the efficacy of allergy immunotherapy.     

Conformational preferences of a short Aib/Ala‐based water‐soluble peptide as a function of temperature

The amino acid Aib predisposes a peptide to be helical with context‐dependent preference for either 3₁₀‐ or α‐ or a mixed helical conformation. Short peptides also show an inherent tendency to be unfolded. To characterize helical and unfolded states adopted by water‐soluble Aib‐containing peptides, the conformational preference of Ac‐Ala‐Aib‐Ala‐Lys‐Ala‐Aib‐Lys‐Ala‐Lys‐Ala‐Aib‐Tyr‐NH₂ was determined by CD, NMR and MD simulations as a function of temperature. Temperature‐dependent CD data indicated the contribution of two major components, each an admixture of helical and extended/polyproline II structures. Both right‐ and left‐handed helical conformations were detected from deconvolution of CD data and ¹³C NMR experiments. The presence of a helical backbone, more pronounced at the N‐terminal, and a temperature‐induced shift in α‐helix/3₁₀‐helix equilibrium, more pronounced at the C‐terminal, emerged from NMR data. Starting from polyproline II, the N‐terminal of the peptide folded into a helical backbone in MD simulations within 5 ns at 60°C. Longer simulations showed a mixed‐helical backbone to be stable over the entire peptide at 5°C while at 60°C the mixed‐helix was either stable at the N‐terminus or occurred in short stretches through out the peptide, along with a significant population of polyproline II. Our results point towards conformational heterogeneity of water‐soluble Aib‐based peptide helices and the associated subtleties. The problem of analyzing CD and NMR data of both left‐ and right‐handed helices are discussed, especially the validity of the ellipticity ratio [θ]₂₂₂/[θ]₂₀₇, as a reporter of α‐/3₁₀‐ population ratio, in right‐ and left‐handed helical mixtures. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Recombinant expression of bioactive peptide lunasin in Escherichia coli

Lunasin, a cancer-preventive peptide, was isolated from soybean, barley, and wheat. Previous studies showed that this 43-amino acid peptide has the ability to suppress chemical carcinogen-induced transformation in mammalian cells and skin carcinogenesis in mice. In this study, we attempted to use the Escherichia coli T7 expression system for expression of lunasin. The lunasin gene was synthesized by overlapping extension polymerase chain reaction and expressed in E. coli BL21(DE3) with the use of vector pET29a. The recombinant lunasin containing his-tag at the C-terminus was expressed in soluble form which could be purified by immobilized metal affinity chromatography. After 4 h, the expression level is above 4.73 mg of recombinant his-tagged lunasin/L of Luria–Bertani broth. It does not affect the bacterial growth and expression levels. This is the first study that successfully uses E. coli as a host to produce valuable bioactive lunasin. The result of in vitro bioassay showed that the purified recombinant lunasin can inhibit histone acetylation. Recombinant lunasin also inhibits the release of pro-inflammatory cytokines (tumor necrosis factor-α, interleukin-1β, and nitric oxide production). Compared with other research methods on extraction or chemical synthesis to produce lunasin, our method is very efficient in saving time and cost. In the future, it could be applied in medicine and structure–function determination.     

Complementary Roles in Cancer Prevention: Protease Inhibitor Makes the Cancer Preventive Peptide Lunasin Bioavailable

Background

The lower incidence of breast cancer among Asian women compared with Western countries has been partly attributed to soy in the Asian diet, leading to efforts to identify the bioactive components that are responsible. Soy Bowman Birk Inhibitor Concentrate (BBIC) is a known cancer preventive agent now in human clinical trials.

Methodology/Principal Findings

The objectives of this work are to establish the presence and delineate the in vitro activity of lunasin and BBI found in BBIC, and study their bioavailability after oral administration to mice and rats. We report that lunasin and BBI are the two main bioactive ingredients of BBIC based on inhibition of foci formation, lunasin being more efficacious than BBI on an equimolar basis. BBI and soy Kunitz Trypsin Inhibitor protect lunasin from in vitro digestion with pancreatin. Oral administration of ³H-labeled lunasin with lunasin-enriched soy results in 30% of the peptide reaching target tissues in an intact and bioactive form. In a xenograft model of nude mice transplanted with human breast cancer MDA-MB-231 cells, intraperitoneal injections of lunasin, at 20 mg/kg and 4 mg/kg body weight, decrease tumor incidence by 49% and 33%, respectively, compared with the vehicle-treated group. In contrast, injection with BBI at 20 mg/kg body weight shows no effect on tumor incidence. Tumor generation is significantly reduced with the two doses of lunasin, while BBI is ineffective. Lunasin inhibits cell proliferation and induces cell death in the breast tumor sections.

Conclusions/Significance

We conclude that lunasin is actually the bioactive cancer preventive agent in BBIC, and BBI simply protects lunasin from digestion when soybean and other seed foods are eaten by humans.     

Dynamic relationships among type IIa bacteriocins: temperature effects on antimicrobial activity and on structure of the C-terminal amphipathic alpha helix as a receptor-binding region

Dynamic aspects of structural relationships among class IIa bacteriocins, which are antimicrobial peptides from lactic acid bacteria (LAB), have been examined by use of circular dichroism (CD), molecular dynamics (MD) simulations, and activity testing. Pediocin PA-1 is a potent class IIa bacteriocin, which contains a second C-terminal disulfide bond in addition to the highly conserved N-terminal disulfide bond. A mutant of pediocin PA-1, ped[M31Nle], wherein the replacement of methionine by norleucine (Nle) gives enhanced stability toward aerobic oxidation, was synthesized by solid-phase peptide synthesis to study the activity of the peptide in relation to its structure. The secondary structural analysis from CD spectra of ped[M31Nle], carnobacteriocin B2 (cbn B2), and leucocin A (leuA) at different temperatures suggests that the alpha-helical region of these peptides is important for target recognition and activity. Using molecular modeling and dynamic simulations, complete models of pediocin PA-1, enterocin P, sakacin P, and curvacin A in 2,2,2-trifluoroethanol (TFE) were generated to compare structural relationships among this class of bacteriocins. Their high sequence similarity allows for the use of homology modeling techniques. Starting from homology models based on solution structures of leuA (PDB code 1CW6) and cbnB2 (PDB code 1CW5), results of 2-4 ns MD simulations in TFE and water at 298 and 313 K are reported. The results indicate that these peptides have a common helical C-terminal domain in TFE but a more variable beta sheet or coiled N terminus. At elevated temperatures, pediocin PA-1 maintains its overall structure, whereas peptides without the second C-terminal disulfide bond, such as enterocin P, sakacin P, curvacin A, leuA, and cbnB2 experience partial disruption of the helical section. Pediocin PA-1 and ped[M31Nle] were found to be equally active at different temperatures, whereas the other peptides that lack the second C-terminal disulfide bond are 30-50 times less antimicrobially potent at 310 K (37 degrees C) than at 298 K (25 degrees C). These results indicate that the structural changes in the helical region observed at elevated temperatures account for the loss of activity of these peptides. The presence of C-terminal hydrophobic residues on one side of the amphipathic helix in class IIa bacteriocins is an important feature for receptor recognition and specificity toward particular organisms. This study assists in the understanding of structure-activity relationships in type IIa bacteriocins and demonstrates the importance of the conserved C-terminal amphipathic alpha helix for activity.     

A disulfide linked model of the complement protein C8γ complexed with C8α indel peptide

In a recent study C8γ (complement protein) with Cys40Ala substitution and a C8α derived peptide with Cys164Ala substitution were co-crystallized and their binding mode was revealed. Computer modeling and molecular dynamics simulations were performed in order to check the hypothesis that the residues Ala164 of C8α and Ala40 of C8γ occupied the right position if cysteine residues were in their place for disulfide bonding. Substitution of these two alanine residues with cysteine along with disulfide bond creation via molecular modeling and subsequent molecular dynamics simulation of the complex corroborated the hypothesis, which was also confirmed from recent crystallographic data. Average RMSD between backbone atoms of the indel peptide during the MD trajectory in comparison with the corresponding sequence of crystal structure of the C8α/γ complex was found only 0.085 nm. Figure Modeling the C*y/α comlexation. Ribbon representation of the C8y complexed with C8α indel peptide initial (green/cyan) X-ray structure and the final MD conformation (magenta/orange) after imposing the disulfide link. Average RMSD between backbone atoms of the indel peptide during MD trajectory in comparison with the corresponding sequence of crystal structure of the C8α/y complex was found only 0.085nm. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effects of guanidine hydrochloride on the conformation and enzyme activity of streptomycin adenylyltransferase monitored by circular dichroism and fluorescence spectroscopy

Equilibrium denaturation of streptomycin adenylyltransferase (SMATase) has been studied by CD spectroscopy, fluorescence emission spectroscopy, and binding of the hydrophobic dye 1-anilino-8-naphthalene sulfonic acid (ANS). Far-UV CD spectra show retention of 90% native-like secondary structure at 0.5 M guanidine hydrochloride (GdnHCl). The mean residue ellipticities at 222 nm and enzyme activity plotted against GdnHCl concentration showed loss of about 50 and 75% of secondary structure and 35 and 60% of activity at 0.75 and 1.5 M GdnHCl, respectively. At 6 M GdnHCl, there was loss of secondary structure and activity leading to the formation of GdnHCl-induced unfolded state as evidenced by CD and fluorescence spectroscopy as well as by measuring enzymatic activity. The denaturant-mediated decrease in fluorescence intensity and 5 nm red shift of λ_max point to gradual unfolding of SMATase when GdnHCl is added up from 0.5 M to a maximum of 6 M. Decreasing of ANS binding and red shift (∼5 nm) were observed in this state compared to the native folded state, indicating the partial destruction of surface hydrophobic patches of the protein molecule on denaturation. Disruption of disulfide bonds in the protein resulted in sharp decrease in surface hydrophobicity of the protein, indicating that the surface hydrophobic patches are held by disulfide bonds even in the GdnHCl denatured state. Acrylamide and potassium iodide quenching of the intrinsic tryptophan fluorescence of SMATase showed that the native protein is in folded conformation with majority of the tryptophan residues exposed to the solvent, and about 20% of them are in negatively charged environment. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 11, pp. 1514–1523.     

Structural recognition of an ICAM-1 peptide by its receptor on the surface of T cells: conformational studies of cyclo (1, 12)-Pen-Pro-Arg-Gly-Gly-Ser-Val-Leu-Val-Thr-Gly-Cys-OH.

The purpose of this study is to elucidate the solution conformation of cyclic peptide 1 (cIBR), cyclo (1, 12)-Pen1-Pro2-Arg3-Gly4-Gly5-Ser6-Val7-Leu8-V al9-Thr10-Gly11-Cys12-OH, using NMR, circular dichroism (CD) and molecular dynamics (MD) simulation experiments. cIBR peptide (1), which is derived from the sequence of intercellular adhesion molecule-1 (ICAM-1, CD54), inhibits homotypic T-cell adhesion in vitro. The peptide hinders T-cell adhesion by inhibiting the leukocyte function-associated antigen-1 (LFA-1, CD11a/CD18) interaction with ICAM-1. Furthermore, Molt-3 T cells bind and internalize this peptide via cell surface receptors such as LFA-1. Peptide internalization by the LFA-1 receptor is one possible mechanism of inhibition of T-cell adhesion. The recognition of the peptide by LFA-1 is due to its sequence and conformation; therefore, this study can provide a better understanding for the conformational requirement of peptide-receptor interactions. The solution structure of 1 was determined using NMR, CD and MD simulation in aqueous solution. NMR showed a major and a minor conformer due to the presence of cis/trans isomerization at the X-Pro peptide bond. Because the contribution of the minor conformer is very small, this work is focused only on the major conformer. In solution, the major conformer shows a trans-configuration at the Pen1-Pro2 peptide bond as determined by HMQC NMR. The major conformer shows possible beta-turns at Pro2-Arg3-Gly4-Gly5, Gly5-Ser6-Val7-Leu8, and Val9-Thr10-Gly11-Cys12. The first beta-turn is supported by the ROE connectivities between the NH of Gly4 and the NH of Gly5. The connectivities between the NH of Ser6 and the NH of Val7, followed by the interaction between the amide protons of Val7 and Leu8, support the presence of the second beta-turn. Furthermore, the presence of a beta-turn at Val9-Thr10-Gly11-Cys12 is supported by the NH-NH connectivities between Thr10 and Gly11 and between Gly11 and Cys12. The propensity to form a type I beta-turn structure is also supported by CD spectral analysis. The cIBR peptide (1) shows structural similarity at residues Pro2 to Val7 with the same sequence in the X-ray structure of D1-domain of ICAM-1. The conformation of Pro2 to Val7 in this peptide may be important for its binding selectivity to the LFA-1 receptor.     

Atomistic probing of aptameric binding of CD19 outer membrane domain reveals an “aptamer walking” mechanism

We propose an integrated structural approach to search potential aptamer molecules for targeting cancer receptor proteins. We used the outer cellular domain of the B-lymphocyte antigen, CD19, as the target for this study. First, using available protein-aptamer structures deposited in the protein data bank as resources, structural annotation was performed to seek the most probable binding aptamer and its potential initial configuration to the CD19 structure. Using this initial structure, molecular dynamics (MD) simulations were performed for adjustment of the aptamer-binding. During this process, we observed an “aptamer walking” mechanism of the binding of the single-stranded RNA-aptamer to CD19: the aptamer molecule gradually adjusts its configurations and shifts toward favorable binding positions. However, the target molecule CD19 maintained a relatively stable conformation during this process. The interface area between the RNA-aptamer and CD19 increased from less than 8 nm² to over 12 nm² during a 2-μs MD simulation. Using a stable binding pose as the starting structure, we manually mutated the RNA-aptamer to a DNA-aptamer and found that the interface area was further increased to over 16 nm², indicating a stronger affinity compared to the RNA-aptamer. The RNA- and DNA-aptamers and their stable binding-poses to the CD19 molecule may be used as templates in designing potential aptamer molecules that target the B-cell marker molecule CD19 with enhanced specificity and stability.     

Effect of Disulfide Bond Incorporation on the Structure and Activity of Endostatin Peptide

The structure and function of a 27-a.a. fragment of the N-terminal sequence of human endostatin (ES-Zn) were compared to those of the mutant peptide (ES-SSZn) obtained by adding Cys-Pro-Ala to the endostatin N-terminus and substituting Asn16 for Cys ensuring formation of a disulfide bond. Structural comparison of ES-Zn and ES-SSZn by far-UV circular dichroism (CD), intrinsic fluorescence, and molecular dynamics simulation methods revealed significant structural perturbations in ES-SSZn, such as elimination of the β-sheet conformer, modification of the N-terminal loop structure, and reorganization of dynamic properties of the entire peptide backbone. ES-SSZn was approximately 2 and 3 times less efficient than ES-Zn and the full-length human endostatin, respectively, in the induction of caspase-3-dependent apoptosis in human umbilical vein endothelial cells (HUVECs) in vitro (p < 0.05). In contrast, treatment of metastatic 4T1 breast tumors in mice with ES-Zn and ES-SSZn (5 mg/kg body weight daily) for 14 days resulted in similar regression of tumor size, comparable downregulation of angiogenesis (CD31 and CD34) and cell proliferation (Ki67), and therefore, the same extent of apoptosis induction (TUNEL, p53, and Bcl-2) for both peptides (as compared to the untreated controls). Western blot analysis of HUVEC and 4T1 tumor lysates revealed the same levels of suppression of key signaling mediators Akt and ERK1/2 by ES-Zn and ES-SSZn. Contrary to the earlier studies, our results showed that the function of the 1-27 endo-statin fragment is independent of its overall structure. Stabilization of the N-terminal loop structure by the disulfide bond incorporation causes relief from structural deviations.     

Biochemical evaluation of a synthesized isoflavone-selenium complex by molecular spectra

A coordination compound of 5, 7-dihydrox-4'-methoxyisoflavone and selenium was synthesized and its structure was identified by IR, LC-MS and (1)H-NMR. Its biochemical effects were investigated using bovine serum albumin (BSA) as a target protein molecule, in which process three-dimensional (3D) fluorescence spectra, ultraviolet spectra, circular dichroism (CD) spectra and fluorescence probe techniques were employed. The interaction of SEIF and BSA was discussed by fluorescence quenching method and F?rster non-radiation energy transfer theory. The thermodynamic parameters ΔH (θ), ΔG (θ), ΔS (θ) at different temperatures were calculated according to Van't Hoff isobaric equation and the results indicated the interaction was an exothermic as well as a spontaneous process. The binding site was explored by fluorescence probe method using warfarin and ibuprofen as markers. Intramolecular forces which are responsible for maintaining the binding were mainly hydrogen bond and van der Waals power. The average distance from the tryptophan residue in domain II of BSA (donor) to SEIF (acceptor) is 3.57 nm at body temperature. The conformation changes of BSA were investigated by 3D fluorescence and CD spectra.     

Molecular dynamics simulation to investigate the impact of disulfide bond formation on conformational stability of chicken cystatin I66Q mutant

Chicken cystatin (cC) mutant I66Q is located in the hydrophobic core of the protein and increases the propensity for amyloid formation. Here, we demonstrate that under physiological conditions, the replacement of Ile with the Gln in the I66Q mutant increases the susceptibility for the disulfide bond Cys71–Cys81 to be reduced when compared to the wild type (WT) cC. Molecular dynamics (MD) simulations under conditions favoring cC amyloid fibril formation are in agreement with the experimental results. MD simulations were also performed to investigate the impact of disrupting the Cys71–Cys81 disulfide bond on the conformational stability of cC at the atomic level, and highlighted major disruption to the cC appendant structure. Domain swapping and extensive unfolding has been proposed as one of the possible mechanisms initiating amyloid fibril formation by cystatin. Our in silico studies suggest that disulfide bond formation between residues Cys95 and Cys115 is necessary to maintain conformational stability of the I66Q mutant following breakage of the Cys71–Cys81 disulfide bridge. Subsequent breakage of disulfide bond Cys95–Cys115 resulted in large structural destabilization of the I66Q mutant, which increased the α–β interface distance and expanded the hydrophobic core. These experimental and computational studies provide molecular-level insight into the relationship between disulfide bond formation and progressive unfolding of amyloidogenic cC mutant I66Q.

An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:23